MODULAR ANALYTICS E combinations 4 Test Principles

Table of contents

Test Principles

In this chapter F Chapter 4

Competitive Test Principle ...............................................................................14
Competitive Principle .................................................................................14
Sandwich Principle ...........................................................................................16
Bridging Principle .............................................................................................18

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Competitive Test Principle

Competitive Test Principle

3 test principles are available on the MODULAR ANALYTICS E combinations
System: Competitive principle for extremely small analytes, sandwich
principle (1 or 2 steps) for larger analytes and a bridging principle to detect
antibodies in the sample.

Competitive Principle
This principle is applied to analytes of low molecular weight, such as FT3.
o In the first step, sample and a specific anti-T3 antibody labeled with a
ruthenium complex are combined in an assay cup.
o After the first incubation, biotinylated T3 and streptavidin-coated
paramagnetic microparticles are added. The still free binding sites of the
labeled antibody become occupied, with formation of an antibody-hapten
complex. The entire complex is bound to the microparticle via interaction
of biotin and streptavidin.
o After the second incubation, the reaction mixture containing the immune
complexes is transported into the measuring cell. The immune complexes
are magnetically entrapped on the working electrode, but unbound
reagent and sample are washed away by ProCell M.
o In the ECL reaction, the conjugate is a ruthenium based derivative and the
chemiluminescent reaction is electrically stimulated to produce light. The
amount of light produced is indirectly proportional to the amount of
antigen in the patient sample.
Evaluation and calculation of concentration of the antigen are carried out by
means of a calibration curve that was established using standards of known
antigen concentration.

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Competitive Test Principle

COMPETITIVE PRINCIPLE

FIRST REACTION

SECOND REACTION

LIGHT REACTION
SIGNAL (LIGHT)

TPA ä ECL

CONCENTRATION
MAGNETIC FORCE &
ELECTRICAL POTENTIAL

ANTIGEN RUTHENIUM LABELLED

ANTIBODY TPA TRIPROPYLAMINE

BIOTINYLATED STREPTAVIDIN-COATED
ANTIGEN MICROPARTICLE

Figure 7 Competitive principle

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Sandwich Principle

Sandwich Principle
The sandwich principle is applied to higher molecular weight analytes, such as
thyroid-stimulating hormone (TSH).
o In the first step, patient sample is combined with a reagent containing
biotinylated TSH antibody and a ruthenium-labeled TSH-specific
antibody in an assay cup. During a nine-minute incubation step,
antibodies capture the TSH present in the sample.
o In the second step, streptavidin-coated paramagnetic microparticles are
added. During a second nine-minute incubation, the biotinylated
antibody attaches to the streptavidin-coated surface of the microparticles.
o After the second incubation, the reaction mixture containing the immune
complexes is transported into the measuring cell; the immune complexes
are magnetically entrapped on the working electrode, but unbound
reagent and sample are washed away by Procell M.
o In the ECL reaction, the conjugate is a ruthenium based derivative and the
chemiluminescent reaction is electrically stimulated to produce light. The
amount of light produced is directly proportional to the amount of TSH in
the sample.
Evaluation and calculation of concentration of the antigen or analyte are
carried out by means of a calibration curve that was established using
standards of known antigen concentration.

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Sandwich Principle

SANDWICH PRINCIPLE

FIRST IMMUNOLOGICAL REACTION

SERUM CONSTITUENTS

SECOND REACTION

LIGHT REACTION
SIGNAL (LIGHT)

TPA ä ECL

CONCENTRATION
MAGNETIC FORCE &
ELECTRICAL POTENTIAL

RUTHENIUM LABELLED
ANTIGEN
ANTIBODY TPA TRIPROPYLAMINE

BIOTINYLATED STREPTAVIDIN-COATED
ANTIBODY MICROPARTICLE

Figure 8 Sandwich principle

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Bridging Principle

Bridging Principle
The bridging principle is similar to the sandwich principle, except that the
assay is designed to detect antibodies, not antigens, (e.g., IgG, IgM and IgA).
This is accomplished by including biotinylated and ruthenium-labeled
antigens in the reagents for which the targeted antibody has affinity.
o In the first step, serum antibodies bind with the biotinylated and
ruthenium-labeled antigens to form an immune complex.
o The immune complex then reacts with streptavidin-coated microparticles
via the biotinylated antigen.
o After the second incubation, the reaction mixture containing the immune
complexes is transported into the measuring cell; the immune complexes
are magnetically entrapped on the working electrode, but unbound
reagent and sample are washed away by Procell M.
o In the ECL reaction, the conjugate is a ruthenium based derivative and the
chemiluminescent reaction is electrically stimulated to produce light. The
amount of light produced is directly proportional to the amount of analyte
in the sample.
Evaluation and calculation of the concentration of the antibody are carried
out by means of a calibration curve that was established using standards of
known antibody concentrations.

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Bridging Principle

BRIDGING PRINCIPLE

FIRST REACTION

SERUM
CONSTITUENTS

SECOND REACTION

LIGHT REACTION
SIGNAL (LIGHT)

TPA ä ECL

CONCENTRATION
MAGNETIC FORCE &
ELECTRICAL POTENTIAL

BIOTINYLATED
ANTIGEN SERUM
TPA TRIPROPYLAMINE

RUTHENIUM ANTIBODIES STREPTAVIDIN-

LABELLED COATED
ANTIGEN MICROPARTICLES

Figure 9 Bridging principle

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Bridging Principle

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